Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/52—Cytokines; Lymphokines; Interferons
  • C07K14/54—Interleukins [IL]
  • C07K14/5443—IL-15
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/19—Cytokines; Lymphokines; Interferons
  • A61K38/20—Interleukins [IL]
  • A61K38/2086—IL-13 to IL-16
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
  • A61P17/04—Antipruritics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
  • A61P17/06—Antipsoriatics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
  • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
  • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• the present invention relates to the branch of molecular pharmacology and in particular refers to a peptide derived from lnlerlcuc ⁇ na-15 (IL 15), which prevents the binding of the atocin to the alpha subunit of the receptor (IL-15R ⁇ ) Therefore, it is useful for the treatment of diseases related to the abnormal expression of IL-15 and / or IL-15Ru in the course of the disease
• the atocin known as IL-15 is a 14-15 kDa ghcoproteln, which was simultaneously identified by two groups as a factor that activates T cells (Grabstein, KH et al, Science 1994, 264 965-968, Burton, JD et al , Proc Nati Acad Sa USA 1994, 91, 4935-4939)
• the messenger ribonucleic acid (mRNA) of i -) Ia IL-15 is present in a wide variety of cells and tissues, however the protein is rarely found in the supernatant of culture of these cells that express the transcript, since there is a strong post-transcriptional control of their expression at the level of translation and intracellular traffic (Bamford RN et al, J Immunol 1998, 160 4418-4426, Kurys G, et al, J Biol Chem 2000, 275 30653-
• IL-15 can exist in active form as a membrane protein (Musso et al Blood 1999, 93 3531-3539) and more recently it was observed that it can function as a ligand or as a receptor for IL -15 expressed as an integral membrane protein acts as a receptor and by binding the alpha subunit of the soluble receptor induces the secretion of the atokines
• the ⁇ subunit of the receptor is shared with IL-2, atocin with which IL-15 has a high structural homology, and the ⁇ subunit is shared with other atokines, such as IL-2, IL-4 , IL-7, IL-9, IL-21 IL-15R (x, is specific for IL-15, to which it binds with very high affinity (Kd 10 ⁇ 11 ) and can be found as a membrane receptor or in soluble form (Budagian V et al J Biol Chem 2004 24 40368-75, Mortier et al, J of
• IL-15 expression associated with the pathogenesis of autoimmune and inflammatory diseases have been found, such as Crohn's disease (Kirman I, Am J Gastroenterol 1996, 91 1789-1794), sonasis (Ruckert R. J Immunol 2000, 165 2240-2250), leukemias (Yamada and Leukemia and Limphoma 1999, 35 37-45) and rheumatoid arthritis (RA) (Mclnnes IB, Immunology Today 1998, 19 75-79)
• RA rheumatoid arthritis
• IL-15 precedes the Tumor Necrosis Factor ⁇ (TNF ⁇ ) in the atokine cascade, proposing a mechanism dependent on cell contact in which IL-15 activated T cells induce the synthesis of TNFu by macrophages.
• TNF ⁇ Tumor Necrosis Factor ⁇
• IL-15 acts as an important factor in the migration of T cells to synovial fluid (Mclnnes et al, Nat Med 1997, 3 189-195) Ziolkowska and collaborators reported that IL-15 induces the expression of IL-17 in the articulation of patients with RA and it is known that this atocin stimulates the release by synovio ⁇ of mediators of inflammation such as IL-6, IL-8 Colony Stimulation Factor of Granulo ⁇ tos-Macrophages, and prostaglandin E2, suggesting an important role to IL-15 in the pathogenesis of RA (Ziolkowska et al, J Immunology 2000,164 2832-2838) Recently, it was shown that IL -15 exacerbates collagen-induced arthritis (collagen mduced arthritis, abbreviated CIA) in a transgenic mouse of this atocin (Yoshihara et al, Eur J Immunol 2007, 37 2744-2752) All these elements suggest that the action of an antagonist of
• WO03017935 it describes four antibodies Two of them called 146B7 and 146H5 that bind to IL-15 in the region that interacts with the ⁇ subunit of the receptor, and inhibit proliferation cell induced by IL-15 of the CT cell line LL-2 and in peripheral blood mononuclear cells (pe ⁇ pheral blood mononuclear cells, abbreviated PBMC), and antibodies called 404A8 and 404E4 that do not inhibit the proliferation of these antibodies 146B7, is in Clinical Phase II for the treatment of The AR by the Amgen company, with the designation of AMG714 Bernard and collaborators in 2004 identified two binding sequences of the IL-15 to the ⁇ subunit of the receptor.
• PBMC peripheral blood mononuclear cells
• sequences are comprised between amino acid (aa) 44 to 52 and 64 at 68 of mature piotein, and describe muteins that can act as agonists or antagonists of IL-15 (Bernard J et al J Biol Chem 2004, 279 24313-24322) Santos et al. Described an IL-15 antagonist peptide (Patent application No. WO2006 / 029578)
• a small-sized peptide (10 aa) as an IL-15 antagonist has the advantage of selectively blocking the binding of the IL -15 to the ⁇ subunit of the receptor and mediate or prevent the effects of IL-15 s due to the interaction with said receptor subunit
• This invention contributes to solving the aforementioned problem by providing a more soluble and more active peptide than the peptide described in Patent Application No. WO2006 / 029578, obtaining a decrease in the inhibitory concentration 50, that is in the concentration of the substance that It produces 50% of 5 inhibition (IC 50 ), from 130 ⁇ M to 8 ⁇ M, due to the change of the second Lys by the amino acid Thr and the obtaining of the dimic form Said sequence (SEQ ID NO 12), synthesized as a linear peptide of 10 amino acids, it interacts with the u subunit of the receptor and shows antagonistic capacity of the IL-15.
• the optimization consisted of making specific substitutions of the 0 amino acids of said peptide to identify the essential amino acids in its antagonistic activity of the IL-15.
• the substitutions that contemplate changes of charge in the particular case of the second hsin of said peptide by threonine with a neutral charge or by Glutamic acid that provides a negative charge, an increase in the antagonistic activity of the peptide 10 times higher was obtained5
• the dimic form of this peptide formed between two peptide molecules through the free cistern is 7 times more active than form monomé ⁇ ca
• the resulting peptide which constitutes the essence of this invention, has the amino acid sequence that appears as SEQ ID NO 12 in the sequence listing East increase in the activity of the peptide as a result of the indicated change of the amino acid Lys by Thr is surprising On the basis of the available information, for people versed in this field of the technique it was not expected that said change in the primary structure of the peptide would generate a marked increase in the biological activity of the peptide, as demonstrated in the embodiments of the present invention
• the peptide with the amino acid sequence listed as SEQ ID No. 12 when binding to the soluble alpha chain, as described in the present invention, can inhibit the effect of reverse signaling through the membrane IL-15 referred to by Budalgian et al. 2004 (Budalgian et al, J Biol Chem 2004, 40
• the present invention contemplates the use of said peptide in the treatment of RA, alone or in combination with some other appropriate molecule, such as, for example, anti-inflammatory spheroidal drugs (such as corticosteroids) and drugs modifying the course of the disease (for example , methotrexate) 0
• an appropriate molecule such as, for example, anti-inflammatory spheroidal drugs (such as corticosteroids) and drugs modifying the course of the disease (for example , methotrexate) 0
• the subject of this invention is also the topical use of this peptide in the treatment of diseases manifested in the skin and in whose lesions the expression of IL-15 is detected in the course of the disease, as in the so ⁇ asis and the cutaneous T-cell lymphoma
• the peptide is used to inhibit the binding of the 5 subunit of the soluble receptor to the IL-15 expressed in the tumor cell membrane and inhibit its migration
• the peptide object of the present invention may have a linear structure or may be dimensioned, and is characterized essentially by its antagonistic capacity of IL-15
• the m vitro effect produced by the peptide or object of the present invention is demonstrated.
• alanine mapping of the peptide described in patent application No. WO2006 / 029578 was performed every mutant peptide was chemically synthesized by the solid phase synthesis method.
• the peptide object of the present invention inhibits the expression of IL-8 induced by IL-15Rc / This same peptide inhibits the expression of IL-6 and the release of TNF alpha (TNFu) induced by IL-15Ru
• the peptide is obtained by genetic manipulation or by chemical synthesis.
• the peptide is obtained as a dimer formed between two peptide molecules comprising the amino acid sequence described in the sequence listing as SEQ ID No 12
• the dimer is obtained from two peptide molecules
• the object of the present invention is also the deoxyribonucleic acid (DNA) that codes for the peptide whose sequence is SEQ ID No. 12, its expression product is capable of binding to the alpha subunit of the cell receptor of lnterleuc ⁇ na-15 or its fraction soluble, and inhibits the biological activity of Ia
• a vector containing said DNA sequence can be used as an alternative for the expression of the peptide sequence
• IL-15 dependent on the alpha subunit of the lnterleuc ⁇ na-15 cell receptor characterized in that
• the therapeutic pharmaceutical composition comprises the dimethyl peptide.
• the pharmaceutical composition comprises the peptide. monome ⁇ co or dime ⁇ co conjugate, or mixed with pharmaceutically acceptable excipients
• the therapeutic pharmaceutical composition capable of inhibiting the biological activity of the IL-15 dependent on the alpha subunit of the cell receptor of lnterleuc ⁇ na-15 contains the nucleic acid chain that 5 codes for the peptide (SEQ ID No. 12)
• a peptide comprising the amino acid sequence described in the sequence listing as SEQ ID No. 12 for the manufacture of a medicament for the treatment of rheumatoid arthritis, Crohn's disease, the so ⁇ asis and for the treatment of prostate cancer
• FIG. 1 Effect of different concentrations of peptide on the proliferation of the CTLL-2 line induced by IL-15 CTLL-2 cells were incubated with 300 pg / mL of IL-15 in combination with serial dilutions of the peptides. The proliferation was
• FIG. 3 Effect of different concentrations of peptide on the proliferation of the K ⁇ T225 line induced by IL-15
• the K ⁇ T225 cells were incubated with 300 pg / mL 5 of IL-15 in combination with signal dilutions of the peptides. Proliferation was measured using Ia MTT mitochondrial staining
• Figure 4 Effect of the peptide on the proliferation of the CTLL-2 line induced by different concentrations of IL-15 and a fixed concentration of peptide
• concentrations of IL-15 used were 75 pg of IL-15 / ml (1), 150 pg of IL-15 / ml O (2), 300 pg of lL-15 / ml (3)
• FIG. 5 Graph showing the inhibitory effect of the peptide on the induction of the mRNA of the IL-8 ( Figure 5A) and the IL-6 ( Figure 5B) in the cells of the PC-3 cell line by incubation with the IL- 15R ⁇ Figure 6.
• Synovial fluid cells of patients with RA were incubated with IL-15 (100 ng / mL) in combination with a concentration of 65 ⁇ M of the peptide per 48h
• the amount of TNF alpha was measured by ELISA. Data from controls with synovial fluid effusion due to trauma are shown.
• Example 1 Optimization of a peptide of IL-15 that binds to IL-15R ⁇ and inhibits the biological function of IL-15.
• the peptides were synthesized by the Fmoc / tBu strategy in syringes.
• the Fmoc-AM-MBHA ream was used at 0.54 mmol / g and the synthesis protocol was followed with 0 mechanical agitation.
• the peptides were lyophilized and They were characterized by HPLC and mass spectrometry. All peptides were obtained with more than 95% purity and there was correspondence between the mass obtained and that expected for the corresponding amino acid sequence S
• Example 2 Effect of the peptides described on the proliferation of the CTLL-2 and KIT225 cell lines.
• CTLL-2 and K ⁇ T225 lines are dependent on IL-15 and proliferate in the presence of atocin. Molecules that bind to IL-15 and prevent signal transduction at 0 through the receptor inhibit the proliferation of these lines.
• dilutions were made of them in 96-well culture plates (Costar, United States) in a volume of 25 ⁇ L of RPMI medium (Gibco) supplemented with 10% bovine fetal serum (Gibco) CTLL-2 or K ⁇ T225 cells (previously washed) were added at a concentration of 5 x 10 3 cells per well and then allowed incubation for 30 min, then a saturating amount of IL -15 of 300 pg / mL was added to each well
• Ia antagonist ability of peptides varying the concentration of IL-15 at a fixed concentration of peptide of 260 uM was incubated for 72 h at 37 0 C and 5% CO 2
• Ia mitocond ⁇ al MTT was also evaluated ( Cosman et al, Nature 1984, 312 768-771) The MTT is reduced by the mitochondrial dehydrogenase of the living cell to form purple
• the antagonistic capacity of the peptides was also evaluated by varying the concentration of IL-15 to a fixed concentration of peptide.
• H it is observed that the antagonistic effect of the mutant peptide Lys-Thr is dependent on the concentration of IL-15
• Example 3 Competent ELISA to study the ability of the peptides to displace the binding to IL-15Ra.
• the peptides were characterized by the binding to IL-15R to ELISA Briefly, 96-well plates were coated with purified IL-15 in PBS and blocked with 1% bovine albumin in phosphate buffered saline ⁇ from the English Phosphate Buffered Saline, abbreviated PBS) Dilutions of each peptide were added to the wells and the incubation was performed for 1 hour at 37 0 C The plate was washed 0 with PBS-Tween 20 and incubated with IL-15Ra-Fc for 1 hour at 37 0 C washes were repeated and incubated with an anti Fc-human IgG-peroxidase for 1 hour at 37 0 C conjugate washes were repeated and revealed Ia antigen-antibody reaction in the presence of substrate and 3, 3 ', 5, 5Tetramet ⁇ lbenc ⁇ d ⁇ na ( TMB) obtaining the OD reading at 450 nm
• TMB 5Tetramet ⁇ lb
• Example 4 Evaluation of the effect of the peptide (SEQ ID No. 12) on the expression of IL-8 and IL-6 in the prostate cancer line PC-3.
• RNA was isolated by the method of T ⁇ Reagent (Sigma) and analyzed by measurement of 5 OD at 260/280 nm and by agarose gel electrophoresis. Reverse transcription and chain reaction of the pohmerase were performed in real time using Quantitect Reverse Transc ⁇ ption Kit and QuantiTect SYBR Green PCR (QUIAGEN) on a Rotor Gene 6000
• Example 5 Inhibition of the production of TNF alpha induced by IL-15 by dimerized SEQ ID No. 12 peptide in synovial fluid cells! of patients with rheumatoid arthritis (RA).
• synovial fluid of patients with RA after obtaining informed consent, was extracted and incubated with hyaluronidase at 10 ⁇ g per mL of fluid for 45 min at 37 0 C After centrifugation at 1200 revolutions per minute for 10 min. synovial fluid cells In 96-well plate 2X1 O * 5 cells per well were incubated with 50 ⁇ g / mL of
• Example 6 Xenotransplantation model in SCID mice of human psoriasis.
• mice (2-3 months old) were transplanted with a 1.5 cm x 1.5 cm piece of skin from a patient with so ⁇ asis
• mice were randomized into three placebo groups, "0 mice” treated with the peptide SEQ ID No. 12 at a dose of 10 mg / kg of weight and treated with cyclopopna A at a dose of 10 mg / kg, on alternate days for 2 weeks
• mice were sacrificed and a 4mm biopsy was taken from each xenotransplant.
• the biopsies were set at formalin for the paraffin embedding, and they were stained with hematocillin and eosin (H)
• the skin from patients with psoriasis treated with the peptide SEQ ID No. 12 or its dimeric form shows a reduction in the severity of the disease, an important reduction in the thickness of the epidermis, in the number of inflammatory cells and the cyiratinocyte cycle, as well as the degree of parakeratosis of psoriatic lesions.

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• 2008
  • 2008-09-30 CU CU20080184A patent/CU23716A1/es not_active IP Right Cessation
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